Q-Why is efficiency so low? What can be done to increase efficiency?

A- The efficiency is consistent with what's reported in the literature with the same technology. There are several ways one can increase efficiency, for instances, adding antibiotic selection and/or FACS sorting to enrich for transfected cells.

Q- What was the reason to choose exactly Exon 2 (in SNCA gene) for cleavage?

Q- Can this correction be used on no iPSC, e.g. neurons from PD patient?

A- Unlikely. The key advantage of iPSCs is that they can be passage for a number of passages without the loss of genomic integrity and pluripotency. It's nearly impossible to obtain a number of primary neurons from PD patients. Neurons derived from PD patient iPSCs usually contain mixed cell types and are much more difficult to culture than iPSCs.

Q- I’ve had problem is the isolation of single iPS cells. What about that in your experiments?

A- As we discussed in the webinar, we employed three genomic analysis tools (In vitro cleavage assay, TaqMan® SNP genotyping and PGM sequencing) for screening and identifying colonies that are clonal population. Sometimes we had to re-pick and re-screen daughter colonies (that were plated at low density) to identify the right clones.

Q- Could using replication-defective lentivirus used to deliver the TAL plasmids? Could it be more efficient?

A- It's possible. We never tried it since we wanted to generate foot-print free isogenic lines. Lenti could leave unwanted integrations.

Q- Whether GBA mutation is associated with other neural disorders?

A- Besides PD, GBA mutations were also found in subjects with dementia with Lewy bodies.

Q- Did you confirm absence of Sendai virus sequences in the iPSC?

Q- Have you ever encountered differences in TAL activity between the 'workhorse' cell line and your actual cell line used?

A- Due to higher transfection efficiency of the workhorse cell line, higher cleavage activity were detected in the workhorse cell line than in iPSCs. Since our focus was iPSCs, we did not perform detailed genomic analysis on the workhorse cell lines.

Q- Have you tested the TaqMan SNP Assay only on single clones? Would it work with all cells from a well for initial verification of TAL function or would the background be too high?

A- We have only run the TaqMan SNP assay on single clones. It is very likely that the background will be to high for screening transfected pools. For detecting rare events in a cell population, Taqman Mutation Detection Assays using castPCR technology is a better alternative.